denaturation at 94 °C for 1min.; primer pairing with the
DNA strand (35°C for 1 min.) and fragment extension step
of 7 minutes at 72°C for 2 min and after 40 cycles, a last
extension step at 72°C. The PCR products were then run
on a 3.5% superfine resolution (SFR) agarose gel in 1 x
TBE bufferat 80V for 2h. Primer pairs that failed to amplify
any product or that have a faint product under these
conditions were tested under the same three annealing
temperatures with three different MgCl2 concentrations:
1, 2 and 4 mM. The gels were stained with ethidium
bromide and photo-documented under UV light.